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Antibiotics (for culture media)
6-methylpurine (6mp) Stock 7.5 mg/mL Working 7.5 µg/mL in NEFF or SPP Ampicillin (Amp) Ampicillin is a penicillin derivative that inhibits crosslinking of peptidoglycan chains in the cell wall of eubacteria. Cells growing in the presence of ampicillin synthesize weak cell walls, causing them to burst due to the high internal osmotic pressure. Stock 100 mg/mL Working 50-100 ug/mL in LB Materials *Ampicillin powder (250 mg in bottle) *ddH2O *100% EtOH Method (makes 2.5 mL of solution) #Add 1.25 mL of ddH2O to the bottle of ampicillin powder. #Add 1.25 mL of 100% EtOH to the bottle of ampicillin powder. #Mix well by vortexing until ampicillin powder is dissolved in solution. Store at 4 C. Aqueous stock solutions are stable for 2-3 months weeks at 4 C. Blasticidin (Bsr) Blasticidin is a peptidyl nucleoside antibiotic isolated from the bacterium Streptomyces griseochromogenes that inhibits protein synthesis by interfering with the peptide-bond formation in the ribosomal machinery. Resistance to blasticidin is conferred by the blasticidin resistance gene from Bacillus cereus (bsr), which codes for blasticidin-S deaminase. Selection with blasticidin is rapid and postive transformants can be obtained in 3-5 days post-selection. Stock 5 mg/mL Working 80 ug/mL in NEFF or SPP Materials *Blasticidin *ddH2O Method (makes 2.5 mL) #Dissolve 20 mg blasticidin powder in 4 ml of ddH2O. #Aliquot 50 µl into sterile microfuge tubes and store in freezer at -20 C. Aqueous stock solutions are stable for 1-2 weeks at 4 C or for 6-8 weeks at -20 C. Do not subject to freeze-thaw cycles; discard unused portions after thawing. Do not store in a frost-free freezer. pH of the aqueous solution should not exceed 7.0 to prevent inactivation of blasticidin. Avoid alkaline pH. Cisplatin (cpt) Cisplatin crosslinks DNA in several different ways, interfering primarily with cell division by mitosis. The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis when repair proves impossible. Stock 20 mM Working 2 mM in NEFF or SPP Cisplatin is a known carcinogen and is extremely toxic. When making stock solution work in a hood and wear a mask as well as a lab coat to protect yourself. Materials *Cisplatin *DMSO (100%) Method (makes ~3.3 mL) #Add 3.33 mL DMSO directly to the 1 g container of cisplatin. #Vortex briefly to mix well. #Make 50 µl aliquots into sterile microfuge tubes that are covered with foil and store at -20 C. Cisplatin is stable for upto 2 years when stored at 4 C in its powdered form. Stock solutions made in DMSO are stable for 6 months at -20 C. Keep the microfuge tubes covered in aluminum foil as the powder and solution are light-sensitive. Do not aliquot more than 200 µl into microfuge tubes as larger volumes take much longer times to thaw at bench-top. Avoid repeat freeze-thaw cycles. Cycloheximide (chx) Cycloheximide inhibits translation in eukaryotes resulting in cell growth arrest and cell death. It is widely used for selection of chx-resistant strains of yeast, fungi and Tetrahymena, controlled inhibition of protein synthesis for detection of short-lived proteins and super-induction of protein expression. Stock 12.5 mg/mL Working 12.5 ug/mL in NEFF or SPP Cycloheximide is extremely toxic. It is a reproductive toxin so wear gloves when handling powder or solutions. Materials *Cycloheximide *Isopropanol (100%) Methods (makes 10 mL) #Measure out 125 mg of cycloheximide and place in a 15 mL conical tube. #Add 10 mL of 100% isopropanol. #Make 1 mL aliquots into sterile microfuge tube and store at -20 C. Kanamycin (Kan) Kanamycin inhibits protein synthesis by binding to the 30s ribosomal subunit and preventing translocation. Kan-r is usually due to a cytoplasmic aminoglycoside phosphotransferase that inactivates kanamycin by covalently phosphorylating it. Kan-r requires phenotypic expression. Stock 25 mg/mL Working 50-100 ug/mL in LB Materials *Kanamycin Sulfate *ddH2O Method (makes 5 mL) #Measure out 125 mg kanamycin sulfate powder into a 15 mL conical tube. #Add 5 mL of ddH2O. #Filter sterilize using a 0.22 µ'' filter in a syringe into 5 sterile microfuge tubes. #Store in freezer. Paromomycin (pm) ''Neomycin is a structural analog of kanamycin that functions by the same mechanism and is inactivated by the same mechanism. Stock 10 mg/mL Working 50 ug/mL in NEFF; 80 ug/mL in SPP Materials *Paromomycin *ddH2O Method (makes 100 mL) #Dissolve 1 g paromomycin powder in 100 mL of ddH2O. #Filter sterilize using 0.25 µ'' filter in a syringe. #Aliquot 1 mL into sterile microfuge tubes and store in freezer at -20 C. Paclitaxel/taxol (txs) ''Paclitaxel stabilizes microtubules inside the cell and as a result, interferes with the normal breakdown of microtubules during cell division and therefore is a mitotic inhibitor. This prevents cells from dividing normally. Stock 20 mM Working 20-40 µM in NEFF or SPP Materials *Paclitaxel *DMSO (100%) Method (makes ~1.5 mL) #Add 1.47 mL DMSO directly to the 25 mg container of paclitaxel and vortex briefly. #Make 50 µL aliquots into sterile microfuge tubes that are covered with foil and place in -20 C. Taxol solutions in DMSO are stable for up to a year at -20 C if they do not undergo several freeze-thaw cycles. Keep the microfuge tubes covered in aluminum foil as the solution is light-sensitive. Do not aliquot more than 200 µL into microfuge tubes as larger volumes take much longer times to thaw at bench-top. Category:Antibiotics Category:LB Category:SPP Category:NEFF